Abstract: In the last ten years, genome-wide association studies (GWAS) identified several single nucleotide polymorphisms (SNPs) that are associated with type 1 diabetes mellitus. Although studies using patient samples and/or rodent cell lines suggest that some SNPs affect genes that are involved in pancreatic beta cell function and survival, the exact role of these SNPs in human pancreatic beta cell remains controversial and the mechanistic nature of these SNPs are largely unknown. This is mainly due to limitations of current model systems. For example, there is large batch-to-batch variation among primary patient islets and the species difference limits the application of rodent cell lines or animal models to determine the role of these SNPs in human pancreatic beta cell function. Thus, there is an emergent need to establish a novel system to study SNPs using disease relevant cell types. Stepwise differentiation of human embryonic stem cells (hESCs), provides an in vitro system to study human embryonic development, as well as the cells to study the role of genetic factors in human diseases. Here, we propose to use isogenic hESCs carrying the diabetes associated SNPs (D-SNP- hESCs) and isogenic hESCs in which diabetes associated genes are knocked out (D-KO-hESCs) to systematically study the role of these SNPs in human pancreatic beta cell development, function, survival and proliferation in both healthy and disease conditions. Understanding the role of these SNPs will significantly enhance our knowledge of genotype to phenotype in diabetes, which will pave the way to the development of drugs for precise therapy of metabolic diseases. In preliminary studies, we have developed 4 isogenic D-SNP-hESCs and 6 isogenic biallelic and monoallelic D- KO-hESCs lines based on INSGFP/W MEL-1 cells. In addition, we have established an efficient strategy to derive glucose-responsive insulin-secreting cells from hESCs. Moreover, we found that PTPN2-/- and rs2542151T/T insulin-expressing cells show increased cell death upon cytokine exposure. Finally, we performed a high content chemical screening using hESCPTPN2-/--derived cells and identified a Wnt inhibitor to correct PTPN2-/- specific hypersensitivity to cytokine treatment. Here, we propose to systematically study the step-wise differentiation of these isogenic D-SNP-hESCs and D-KO-hESCs, define the function of the insulin-secreting cells derived from wild type (wt), mutant and KO hESCs, and explore the functional, proliferation and survival of the insulin-secreting cells in disease condition. Finally, we will explore the mechanistic nature of these SNPs. The proposed study will not only provide novel insights into the role of SNPs in human pancreatic beta cell generation and function, but also provide a platform to evaluate new methods or drug candidates for precision therapy of diabetes. Toward this goal, the following aims are proposed: Aim 1: Determine the role of SNPs and gene KO in the generation and function of human pancreatic beta cells. Aim 2: Define the role of SNPs and gene KO in human pancreatic beta cell function and survival in disease conditions. Aim 3. Decode the mechanistic nature of SNPs and identify the small molecules rescuing SNPs or mutations specific beta cell dysfunction.